The long-term objective of this project is to understand the role of MelCAM and its complementary ligand in cell-cell adhesion. MelCAM is a recently discovered member of the immunoglobulin superfamily of CAMs, which shows dramatically elevated expression in many melanoma and breast cancer cell lines. Structure-function analysis of MelCAM and its 95 kDa ligand will use protein chemical and molecular biology methods to study both natural proteins from melanoma cells and recombinants expressed in insect cells, E. coli, and stably-transfected human cells. Methods will include: HPLC peptide mapping, N-terminal sequencing, mass spectrometry, microcalorimetry, circular dichroism, immunological methods, and cell binding assays. The functionally-important interactions that will be studied in this project are: (1) lateral association of MelCAM within a single plasma membrane (cis homodimers); (2) heterotypic intercellular binding to a novel 95 kDa ligand, and (3) association with the cytoskeleton. The overall hypothesis is that heterotypic intercellular association with MelCAM with its 95 kDa ligand forms important cell-cell adhesions that play important, but poorly defined, roles in primary tumor growth as well as the invasive stages of metastasis. A specific hypothesis that will be directly tested is that these moderate affinity intercellular interactions are dependent upon the oligomeric state of MelCAM within the cell membrane, i.e., a cis MelCAM homodimer is essential for adhesion. In addition, association of MelCAM with the cytoskeleton does occur and is likely to be involved in intercellular adhesion and/or transmembrane signaling. Signal transduction and/or interaction with the cytoskeleton may involve phosphorylation of the MelCAM cytoplasmic domain. The specific aims of this proposal are: (1) clone and characterize the heterotypic 95 kDa ligand; (2) map and characterize MelCAM binding sites; and (3) identify cytoskeletal proteins associated with MelCAM and define their role. MelCAM expression correlates closely with the transformed phenotype; only trace expression occurs on normal melanocytes in vivo, while very high MelCAM expression is observed in aggressively growing tumors and in most melanoma cell lines. The MelCAM-ligand adhesion system is both essential and sufficient for cell-cell aggregation of melanoma cells. This project will produce a detailed understanding of the structural and functional properties of MelCAM-mediated cell-cell adhesion, which should provide critical insights concerning its importance during tumor progression.